Phenotypic signatures and genetic determinants of oxacillin tolerance in a laboratory mutant of Staphylococcus aureus.

Marilyn Chung,Herminia De Lencastre,Alexander Tomasz,Vitor Borges,João Paulo Gomes

doi:10.1371/journal.pone.0199707

Marilyn Chung, Herminia De Lencastre + Show 3 more

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https://doi.org/10.1371/journal.pone.0199707

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Abstract

Addition of β-lactam antibiotics to growing cultures of bacteria inhibit synthesis of the bacterial cell wall peptidoglycan accompanied by killing (loss of viable titer) and lysis (physical disintegration) of the cells. However, it has also been well established that these antibiotics are not effective in killing non-growing or slow-growing bacteria and the mechanism of this “antibiotic tolerance” is not well understood. In this study, we report on the genetic basis and phenotypic properties of an antibiotic tolerant derivative of the methicillin susceptible S. aureus strain 27s. Cultures were exposed to “pulses” of high concentrations of oxacillin followed by outgrowth of the surviving bacteria. This procedure quickly selected for antibiotic tolerant mutants with an increased ability to survive antibiotic treatment without increase in the MIC value for the antibiotic. Such mutants also exhibited longer lag phase, decreased lysis, virtually no change in antibiotic susceptibilities, cross tolerance to D-cycloserine and vancomycin, and increase in biofilm formation in the presence of high concentrations of oxacillin. Whole genome sequencing showed that these altered properties were linked to mutations in the atl and gdpP genes.

Highlights

Staphylococcus aureus is a major pathogen and methicillin resistant strains of S. aureus (MRSA) are often the primary cause of infections in hospital and clinical settings [1]
In a search for the genetic basis and phenotypes of βlactam tolerance, we isolated a tolerant mutant, designated as 14c5p [i.e., obtained after five passages of a 14th cycled oxacillin mutant (14c)], from the parental strain 27s, which is a derivative of the methicillin susceptible S. aureus (MSSA) strain NCTC8325
The tolerant mutants 14c and 14c5p remained susceptible to all eight antibiotics tested by disk diffusion and Etest

Summary

Introduction

Staphylococcus aureus is a major pathogen and methicillin resistant strains of S. aureus (MRSA) are often the primary cause of infections in hospital and clinical settings [1]. Antibiotics such as beta lactams have been developed to treat such infections. In S. aureus resistance to beta lactams such as methicillin or oxacillin arose through the acquisition of a foreign piece of DNA called the SCCmec cassette, which carries the methicillin resistance determinant mecA encoding the penicillin binding protein (PBP)2A –a key component of the resistance mechanism in most MRSA isolates [2]. Methicillin susceptible S. aureus (MSSA) are pathogenic and can acquire tolerance to β-lactams, i.e., the capacity to survive treatment with antibiotics.

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